Lecture 11 - Hormone analysis Flashcards
3 examples of hormone analysis use in Reproductive endocrinology:
- Determination of ovulation time for mating time
Progesterone (dog), LH
- Pregnancy, nonpregnancy diagnosis
relaxin (dog), oestrone sulphate (cow, goat), progesterone (cow) - Fertility management (dairy cattle)
progesterone
Doping in production animals
- Female animals can be treated with androgens or synthetic androgens (trenbolone acetate) to improve muscle growth and decrease carcass fat.
- Intact males can be treated with estrogenic
compounds to increase carcass fat and meat
tenderness (estradiol, melengestrol acetate)
boar taint
The pheromone androstenone (5α-androst-16-en-3-one) and the tryptophan metabolite skatole are the main factors responsible for
characteristic off-flavor in male pigs.
Androstenone is produced and secreted by the Leydig cells, together with other steroids. It is lipophilic and accumulates in adipose tissue.
Name 3 Types of hormone assays:
- Bioassays
- Immunoassays
- Chromatography
Explain Bioassay
method for measuring the amount of a hormone is based on measuring the physiological responses caused by the
hormone. The parameter or response should be specific for the hormone.
Material:
* Whole animals (in vivo test),
* Tissue preparations (in vitro test)
the Xenopus test
African clawed frog: Xenopus laevis
This test involved injecting a toad with urine from supposedly pregnant woman.
“positive” test result: an unfertilized female frog laying a bunch of eggs.
hCG can induce the female Xenopus toad to lay hundreds of eggs.
Disadvantages of bioassays (4)
- A lack of sensitivity, particularly with whole animal studies, which require a lot of hormone to produce a response.
- Poor reproducibility due to the wide variability in response that is obtained with different animals.
- High cost and difficulty of use for animals and animal preparations.
- Ethical considerations
Immunoassay uses the basic
immunology concept of
an antigen binding to its specific antibody.
Explain Radioimmunoassay
uses radiolabeled molecules in a stepwise formation of immune complexes. Final step uses machine to measure radioactivity.
Disadvantages of radioimmunoassay:
* Radioactivity
* Short self-life of the isotopes (125I)
* Expensive equipment
Principles of immunoassays for hormone analysis.
Dependent on the size of the hormone to be
measured two types of assay methods are used:
- Sandwich method (big hormones with high molecular mass)
- Competitive assay (haptens (small hormones), molecular mass < 1000 D)
Explain Sandwich method
A principle of immunoassay for hormone analysis. The hormone can be captured by antibodies from both sides which results in a sandwhich.
- Glycoprotein hormones, peptide
hormones (eCG, hCG, LH, FSH, TSH,
STH)
Explain competitive assay
A principle of immunoassay for hormone analysis.
Some hormones are small steroid hormones that cannot be captured by antibodies so in this case competitive methods are used. Labelled hormone competes against non-labelled - the more non labelled hormone present, the higher the likelihood it binds instead of the labelled resulting in deeper or lighter color (line) detected.
- steroids (progesterone, cortisol, estradiol),
thyroid hormones (T4, T3)
Components of immunoassays (5)
- Hormone specific antibodies
- Label (labelled hormone or labelled hormone specific antibody)
- Separation system
- Calibrators
- Quality control samples
How’re monoclonal antibodies produced?
Continuous cultures of fused cells secreting antibody of predefined specificity.
Diff. between poly- and monoclonal antibodies.
Polyclonal involve different antibody types: consist of more than one type of clone.
Monoclonal antibodies are all clones.
